Detection reagent kit of fluorescent quantitation PCR latent hepatitis b viruses

A fluorescent quantitative and hepatitis B virus technology, applied in the direction of microbial measurement/inspection, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of HBV base mutation, OBI missed detection, low practicability, etc., and achieve strong specificity , High detection efficiency, efficient and accurate quantitative effect

Pending Publication Date: 2019-06-07
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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Problems solved by technology

[0006] In view of the technical shortcomings of nested PCR detection, the purpose of the present invention is to provide a method and kit for detecting viruses by double-gene simultaneous fluorescence quantitative PCR, which is used to solve the problem of low practicability of OBI clinical detection methods in the prior art, HBV In the event of base mutations, OBI missed detection, false negatives, etc., this method monitors the results in real time without further gel electrophoresis analysis

Method used

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  • Detection reagent kit of fluorescent quantitation PCR latent hepatitis b viruses
  • Detection reagent kit of fluorescent quantitation PCR latent hepatitis b viruses
  • Detection reagent kit of fluorescent quantitation PCR latent hepatitis b viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Sensitivity test of quantitative detection of HBV by fluorescence quantitative PCR of single-plex S gene region

[0115] 1. Dissolve the standard positive template with distilled water and dilute it to 3×10 5 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL, 3×10 1 copies / μL, 3×10 0 copies / μL.

[0116] 2. Fluorescent quantitative PCR experiment (20 μL amplification reaction system): 10.0 μL fluorescent quantitative reaction solution, 0.7 μL (0.5 μM) forward primer of the S gene region primer set, 0.7 μL (0.5 μM) reverse primer of the S gene region primer set , fluorescent probe 1.5 μL (0.2 μM), primer probe set selected from combination S1, standard positive template 7.0 μL.

[0117] Fluorescent quantitative PCR reaction parameters are as follows: pre-denaturation: temperature is 95°C, time is 10min; denaturation: temperature is 95°C, time is 10s, annealing: temperature is 62°C, time is 30s, 45 cycles of denaturation and annealing, this step consist...

Embodiment 2

[0119] Sensitivity test of quantitative detection of HBV by fluorescence quantitative PCR of single C gene region

[0120] Fluorescent quantitative PCR experiment (20 μL amplification reaction system): 10.0 μL fluorescent quantitative reaction solution, 0.7 μL (0.5 μM) forward primer, 0.7 μL (0.5 μM) reverse primer of C gene region primer set, 1.5 μL fluorescent probe ( 0.2 μM), the primer probe set was selected from combination C1, and the standard positive template was 7.0 μL.

[0121] Fluorescent quantitative PCR reaction parameters are as in Example 1, and the results are shown in the attached figure 2 .

[0122] The results showed that the minimum detection concentration of the S and C gene regions of HBV plasmid DNA by single-plex fluorescent quantitative PCR was 3.0copies / uL.

Embodiment 3

[0124] Quantitative detection of HBV by double-gene simultaneous fluorescent quantitative PCR

[0125] Fluorescent quantitative PCR experiment (20 μL amplification reaction system): 10.0 μL fluorescent quantitative reaction solution, 0.25 μL (0.5 μM) forward primer of the S gene region primer set, 0.25 μL (0.5 μM) reverse primer of the S gene region primer set, fluorescent Probe 0.50 μL (0.2 μM), C gene region primer set forward primer 0.25 μL (0.5 μM), C gene region primer set reverse primer 0.25 μL (0.5 μM), fluorescent probe 0.50 μL (0.2 μM), primer The probe sets were selected from combinations S1 and C1 respectively, and the standard positive template was 8.0 μL.

[0126] Fluorescent quantitative PCR reaction parameters are as in Example 1, dual-channel fluorescence simultaneous detection, set as attached image 3 .

[0127] The results of the amplification of the S gene region are shown in the appendix Figure 4 , the results of the amplification of the C gene region ...

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Abstract

The invention provides a detection reagent kit of fluorescent quantitation PCR latent hepatitis b viruses. The detection reagent kit comprises fluorescent quantitation reaction liquid and a primer andprobe combination, wherein the primer and probe combination comprises one or more of an S gene region primer and probe combination, and a C gene region primer and probe combination. Primers and probes are respectively designed for a hepatitis b virus S and a C consensus sequence, a dual dual-gene fluorescent quantitation PCR technique is used for performing same-pipe detection at the same time onHBVDNA. The detection reagent kit is simple and quick to operate, the sensitivity and the specificity of a latent hepatitis b virus detection method are improved, the probability of false negative results caused by mutation can be reduced to the best, and the detection efficiency can be further improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a kit for detecting occult hepatitis B virus by double-gene simultaneous fluorescence quantitative PCR. Background technique [0002] HBV infection remains a major public health problem worldwide. The 2017 World Health Organization (WHO) global hepatitis report estimated that 3.5% (257 million) of the world's population still suffer from chronic hepatitis B virus infection, and the hepatitis B surface antigen (Hepatitis B surface antigen, HBsAg) had the highest positive rate (6.2%). Since 1992, my country has incorporated hepatitis B vaccination into the routine infant immunization program, and the hepatitis B immunization prevention work for newborns has achieved remarkable results. However, the burden of HBV infection in China is still the highest in the world, with 240 million people worldwide. One-third of chronic HBV patients live in China. [0003] Generally, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
Inventor 赵耀杨宇婷
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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